The present invention relates to the methods of producing plant seeds comprising microbes, as well as compositions of plants and seed comprising microbes.
Agriculture faces numerous challenges that are making it increasingly difficult to provide food, materials, and fuels to the world's population. Population growth and changes in diet associated with rising incomes are increasing global food demand, while many key resources for agriculture are becoming increasingly scarce. By 2050, the FAO projects that total food production must increase by 70% to meet the needs of the growing population, a challenge that is exacerbated by numerous factors, including diminishing freshwater resources, increasing competition for arable land, rising energy prices, increasing input costs, and the likely need for crops to adapt to the pressures of a more extreme global climate. The need to grow nearly twice as much food in more uncertain climates is driving a critical need for new innovations.
Today, crop performance is optimized via of technologies directed towards the interplay between crop genotype (e.g., plant breeding, genetically-modified (GM) crops) and its surrounding environment (e.g., fertilizer, synthetic herbicides, pesticides). While these paradigms have assisted in doubling global food production in the past fifty years, yield growth rates have stalled in many major crops and shifts in the climate have been linked to production declines in important crops such as wheat. In addition to their long development and regulatory timelines, public fears of GM-crops and synthetic chemicals has challenged their use in many key crops and countries, resulting in a complete lack of acceptance for GM traits in wheat and the exclusion of GM crops and many synthetic chemistries from European markets. Thus, there is a significant need for innovative, effective, and publically-acceptable approaches to improving the intrinsic yield and resilience of crops to severe stresses.
Like humans, which benefit from a complement of beneficial microbial symbionts, plants have been purported to benefit somewhat from the vast array of bacteria and fungi that live both within and around their tissues to support their health and growth. Endophytes are fungal or bacterial organisms that live within plants. Bacterial and fungal endophytes appear to inhabit various host plant tissues and have been isolated from plant leaves, stems, or roots. There is an increasing appreciation of the roles played by microbes, including endophytic bacteria, in improving plant growth, health and productivity. For example, association with certain microbes can promote plant growth using an array of mechanisms, including playing an essential role in biological nitrogen fixation (BNF), the synthesis of phytohormones and vitamins, increasing the host plant's tolerance to numerous environmental stresses (including both biotic and abiotic stresses), alteration of hormone production, as well as increasing the bioavailability to the plant of macro- and micronutrients such as phosphorus and iron.
Endophytic organisms associated with plants occupy a relatively privileged niche within a plant and contribute to plant health or growth. Endophytes have been targeted as valuable sources of new bioactive compounds. Endophytes inhabit plant tissues, particularly the so-called intercellular space. Endophytic microorganisms have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytic microorganisms can promote plant growth and yield and can act as biocontrol agents. Endophytes can also be beneficial to their host by producing natural products that are beneficial to the plant and could also be harnessed for potential use in medicine, agriculture or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of non-food crops for biomass and biofuel production.
Numerous attempts are known which aimed at improving the establishment of transferring beneficial microbes to another plant: these efforts include the coating of seeds with microbes, application of microbes to the soil, or even direct injection of the microbes into plant stems or leaves, as well as foliar application of microbes. Seed treatment (soaking and embedding in carrier material) with bacterial inocula prior to sowing is one method of inoculation. Peat (carrier based mixing) inoculants have been used to apply microbes to plants; however, several other commercial preparations have been marketed. Crop Genetics International Ltd. developed a seed inoculation technique by applying a pressure differential to infuse the bacterial suspension into imbibed seeds and re-drying the seeds (U.S. Pat. No. 5,415,672 A).
However, these conventional methods suffer from variability stemming from the reduced viability of microbes through the processing of the microbes, including drying, fertilizer contact, seed coat toxicity, incompatible pesticidal and mineral additives, or long periods of storage in varying environmental conditions (e.g., heat, humidity, etc.). Likewise, several soil and environmental stresses affect the survival/colonization efficiency of the inoculant strains. Bacterial population density, the host plant species, agronomic practices and climatic conditions are among the important factors for the success of biological plant colonization.
Moreover, current inoculation methods of microbial colonization of plants with the desired microorganisms have proven difficult and often yield inconsistent results, making it difficult to apply this technology on an industrial scale. For example, microorganisms used in seed coating often do not survive well or are unable to colonize the plant (because the microorganisms on the outside can be hindered in entering the seed or plant). If the plant is mechanically (or otherwise) wounded to provide an entry, this can put the health of the seed, seedlings or plant at risk, because harmful microorganisms could enter the plant as well in an unprotected manner. Moreover, even if the microorganisms can colonize a given plant, there can be a natural loss of viability and the efficiency of colonization can be low. More complex inoculation techniques (e.g., by applying vacuum or pressure infiltration, inoculation by injection, etc.) are also causing risk for the plant and are—most importantly—challenging to transfer to a large scale or industrial applicability and are thus not effective.
Therefore, there is a need to produce plants with improved traits without genetic modification. There is also a need to provide plants and seeds with defined additions to their endophyte populations.